Ansamitocins are highly cytotoxic compounds derived from fermentation of microorganisms such as Actinosynnema pretiosum. Ansamitocins have been chemically converted into thiol-containing maytansinoids, whose therapeutic use in the form of cell binding agent-maytansinoid conjugates has been described (U.S. Pat. Nos. 5,208,020; 5,416,064; 6,333,410; and 6,441,163).
The fermentation process with Actinosynnema spp strains such as Actinosynnema pretiosum produces several ansamitocin species bearing different ester substituents at C-3 (FIG. 1). The various C-3 esters produced include P-3 (iso-butyryl), P-3′ (n-butyryl), P-2 (propionyl), P-4 (iso-valeryl), P-4′ (n-valeryl). All of these esters can be reductively cleaved to give the C-3 alcohol maytansinol (P-0), which is the precursor for the synthesis of thiol-containing maytansinoids. In addition, minor amounts of undesired ansamitocins which are modified at other sites, such as N-demethyl, 20-O-demethyl, and 19-dechloro are produced. Upon reductive de-acylation, these ansamitocins do not produce maytansinol.
Processes for the production of ansamitocin from fermentation of Actinosynnema spp have been described (U.S. Pat. Nos. 4,162,940; 4, 450,234; 4,228,239; 4,331,598; and 4,356,265). The yield of ansamitocins produced varies, with titers generally ranging from 12 mg/L to 100 mg/L. The ansamitocins are typically recovered and purified by a multistep process involving addition of a filter aid and an organic solvent to the whole fermentation broth, followed by concentrating the organic layer and precipitation with petroleum ether. The precipitate was further purified using silica chromatography and crystallization, followed by further purification by recrystallization or chromatography.
Thus, the process is cumbersome and involves several steps where highly toxic material has to be handled. This renders the scale up of such a process very difficult. In addition, the safety of the human operator has to be ensured throughout the various processing steps.
A recent application (US 2002/0015984 A1) claims certain improvements in the process for the production of ansamitocins. The ansamitocin titers in the fermentation broth are reported to have ranged from 65 to 86 mg/L. The claimed improvements included heat inactivation of the broth at 75° C., extraction into an aromatic hydrocarbon solvent such as toluene, chromatography through an open silica column, followed by crystallization. In order to reduce the cost of ansamitocin production, new Actinosynnema spp strains that give significantly higher titers (up to 400 mg/L in fermenters) than those previously described, have been produced. The processes previously described for the production of ansamitocins have several drawbacks, and thus cannot be adapted for the new high producing strains that have been developed. For example, heat inactivation at 75° C. results in some degradation of ansamitocin and a 10 to 20% loss in yield. Extraction of fermentation broth containing high ansamitocin content with aromatic hydrocarbons is inefficient and incomplete, since the ansamitocins are not highly soluble in such solvents. Purification of ansamitocins on open self-packed silica columns has two drawbacks: 1) lot to lot variability in purity and recovery, and 2) significant human exposure resulting in safety concerns. Thus, a need exists to produce ansamitocins in high yields and also to provide an efficient process for its isolation and purification, while minimizing worker exposure to the highly toxic drug.